The enzyme creatine phosphokinase, hereafter abbreviated CPK, is found in normal human serum and catalyzes the transfer of phosphate from creatine phosphate to adenosine diphosphate (ADP) to form adenosine triphosphate (ATP) and creatine. Analysis of CPK concentration in the serum is commonly used in the diagnosis of human diseases or disorders such as myocardial infarction, pulmonary infarction, cerebral infarction, hypothyroidism, liver disorder, skeletal muscle damage due to trauma or injury, and muscular dystrophy.
An assay method for CPK using the reverse reaction of creatine phosphokinase and adenylate kinase was developed by Oliver, Biochem J. 61, 116(1955). This method was later improved by Rosalki, J. Lab. Clin. Med. 61, 696(1967), and Hess, et al., Am. J. Clin. Path 50, 89(1969). Further modifications of the procedure permitted the use of nicotinamide adenine dinucleotide (NAD) instead of nicotinamide adenine dinucleotide phosphate (NADP) in the final ultraviolet detection step. See Okinaka, et al., J. Lab and Clinical Med. 64, 299(1964). The key reactions may be summarized as follows: EQU I. oxidized inactive CPK + reduced thiol activator .fwdarw. reduced active CPK + oxidized thiol activator EQU II. ADP + creatine phosphate .sup.active CPK ATP + creatine EQU III. ATP + glucose .sup.hexakinase ADP + glucose-6-phosphate EQU IV. glucose-6-phosphate + NAD.sup.+ .sup.glucose- 6-phosphate dehydrogenase 6-phosphogluconate + NADH
to eliminate the need for ultraviolet detecting instrumentation the NADH formed in reaction IV above may be converted to NAD.sup.+ with the subsequent reduction of the tetrazolium compound 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride, hereafter INT, to form the red formazan dye INTH via enzymatic catalysis by diaphorase. See Avigad and Levin, N, Europ. J. Biochem 1, 102(1969). This reaction may be expressed as follows: EQU V. NADH + INT .sup.diaphorase INTH + NAD.sup.+
Hereinafter the sequence of reactions I through V will be called the Oliver-Rosalki-formazan method. A body fluid, such as serum, plasma, lymphatic fluid or the like is analyzed for CPK activity using this method by adding test fluid to a prewarmed reconstituted lypholized substrate containing activator, primary substrates, intermediate substrates, final substrates, coupling enzymes, adenylate kinase inhibitor AMP, buffering compounds and stabilizers. The test fluid is incubated (at about 37.degree. C) for a fixed period of time (usually about 10 minutes) and then the reaction is stopped by the addition of acid. Likewise, water is added to another substrate and run as a reagent blank. Control serum having a known CPK activity is used to determine the activity of the unknown test serum.
The Oliver-Rosalki-formazan method as currently in use has four major drawbacks. First, there is variable lag time in the activation of CPK by the thiol activator in reaction I described above. While this lag can be followed using sophisticated continuously monitoring colorimetric measuring equipment having a reagent blank vial and using a double beam, this lag is a disadvantage when an end point method is used as employed in batch analysis. Thus if less sophisticated colorimetric equipment is used for end point method analysis some variation in results is unavoidable in the method as currently employed. Second, there is a continuous increase in the reagent blank absorbance following reconstitution of the substrate because of the presence of all the components necessary for non-serum CPK chormogen production. Third, there is a difference between the reagent blank vial's absorbance and the test vial's absorbance due to the variable degree of solubilization of the final chromogen, INTH, in the presence of serum. This results in an inadequate reagent blank. Fourth, the control serum is unstable and displays variable activation times which necessitates labelling commercial serum containing CPK with wide ranges of activity.
The present invention is directed to an improved Oliver-Rosalki-formazan method which overcomes the disadvantages of the current method. This improved method allows for accurate enzymatic measurement in end point analysis using standard clinical grade colorimeters.